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Drg1 is a human GTPase belonging to the Obg family that is found in archaea and in eukaryotes but not in eubacteria [1, 2]. It was first discovered as a developmentally regulated GTPase, abundantly transcribed in growing cells in mammalian and frog embryos [1, 3, 4] and in actively growing tissues in plants [5, 6], being predicted to regulate cell growth [5, 7]. It associates with polysomes, playing a role in translation .
DRG proteins from nearly all organisms contain 365–370 residues, with several well differentiated domains: the N-terminal HTH domain, the canonical GTP binding domain, the S5D2L insertion domain and a small C-terminal ThrRS, GTPase, SpoT (TGS) domain . The G domain responds to the classical architecture of GTPases with the five G boxes and two switch regions , whereas the TGS domain has a predominantly β-sheet structure sharing a common basic fold similar to ubiquitin and sumo proteins (PDB code 2EKI). The TGS domain appears in other members of the family such as YchF and Obg, and also in threonyl tRNA synthetases, suggesting a regulatory role .
GTPases are typically very inefficient enzymes; thus many of them become associated with specific regulators, the GDP–GTP exchange factors (GEFs) to mediate their activation, and the GTP-hydrolysis activating proteins (GAPs) to terminate their activity . GAPs usually provide a catalytic residue to the active center that functions in trans, while GEFs help to lower the affinity for the guanine nucleotides. Alternatively, GAP-independent ways of activation have been found such as dimerization, association with ribosomal subunits or cation binding, usually a potassium ion . Neither GAPs nor GEFs have been found for Drg1, which according to its sequence has been predicted to be a potassium-dependent GTPase .
Nucleotide cleavage in GTPases is triggered by a water molecule conducting a nucleophilic attack on the gamma phosphate of GTP. There are different ways of positioning or aligning the attacking water molecule and stabilizing the transition state of the phosphoryl transfer reaction . Apart from a catalytic residue generally provided by switch II, the catalytic machinery can be further reinforced by a positive charge introduced by a GAP in trans (usually the so-called arginine finger or asparagine thumb, as in Rho and Ran, respectively ). Alternatively, the same role can be taken by the binding of a monovalent cation, such as potassium ion (chemical GAP), coordinated by switch I. The potassium contribution has recently been unraveled thanks to the solved structures of MnmE and FeoB in their active and inactive states [15, 16]. Surprisingly, even though the main catalytic residue is usually found in a conserved position (Gln61 from Ras) in switch II, a novel group of GTPases with a hydrophobic residue instead, called HAS-GTPases , has been described in which the catalytic residue either has been replaced by a network of backbone contacts, as in FeoB , or is provided by new regions of the protein, such as Glu282 from MnmE .
After sequence alignments and the solved structure of the yeast homolog Rbg1 (PDB code 4A9A), DRG factors can be considered as HAS-GTPases, provided that we observe that instead of the catalytic glutamine there is a hydrophobic residue, Ile122, in switch II adopting a ‘retracted conformation’. On the other hand, although there is considerable conformational variability in the switch regions in the absence of a nucleotide, and they can adopt different conformations according to the crystal packing, the arrangement of switch I resembles that of FeoB or MnmE complexed with GDP [15, 16].
Interestingly, although Drg1 is not essential, two and three highly similar paralog proteins can be found in animals and plants, respectively, suggesting an important role of these redundant proteins in fundamental biological processes [1, 8]. In this sense, microarray data and reporter gene assays have shown that DRGs from Arabidopsis thaliana are transcriptionally regulated, and the protein accumulates directly and specifically in response to heat stress and desiccation [18, 19]. Besides, Drg1 from Candida albicans plays a role in the control of invasive filamentation . Lastly, no clear phenotype has been found for the Drg1 or Drg2 knockdown human cell lines  or for the yeast gene deletion strains . Taken together, these observations suggest that Drg1 may require certain specific conditions to switch on its regulatory function through the protein synthesis machinery.
In this regard, the biological functions of the members of two subfamilies of the Obg family of GTPases which have been better characterized to date: Obg and YchF have been related to stress response and ribosome assembly [23-28].
Drg1 is subjected to a tight regulation. It has been found to be a target of sumoylation, stimulated by the MEKK1 Map3kinase , of ubiquitination [29, 30] and of phosphorylation, undertaken by MPSK1 . The physiological meaning of the latter action is still unclear, but other GTPases are downregulated or upregulated by phosphorylation .
Lerepo4 (or Dfrp1) was first identified in vitro and further confirmed in vivo as a partner of Drg1 [1, 8, 30], and it has been demonstrated in yeast to be responsible for its recruitment to polysomes [9, 33]. Automated predictions have considered Lerepo4 as an intrinsically unstructured protein, and the structure of the C-terminal part of Tma46 solved in complex with Rbg1 corroborates this idea as it adopts a non-globular extended conformation, contacting the GTPase and TGS domains of Rbg1 .
Lerepo4, highly conserved among eukaryotes, has two unique CCCH-type N-terminal zinc fingers and a C-terminal Dfrp domain, being the second responsible for the interaction with Drg1 . The two consecutive zinc fingers of Lerepo4 are similar to the C3HC4-type and C3H2C3-type RING fingers, characteristic of proteins involved in degradation through the ubiquitin proteasome pathway. Lerepo4 is somehow related to this pathway, since it diminishes Drg1 protein degradation by preventing its polyubiquitination , thus representing a way of regulating Drg1 protein levels, which may be important for its function.
Additional roles of Lerepo4 are poorly understood. However, recent data have revealed that human cells increase Lerepo4 expression upon HIV infection , and it is also upregulated in neuronal rat cells by neurotrophic factor stimulation .
To gain further insights into the function of Drg1, the role of the TGS domain, the mode of interaction with Lerepo4 and its effects on GTPase activity, the kinetic characteristics of Drg1 have been addressed in the present work.
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Taken together, our data show that Drg1 is a low efficient GTPase, with abnormal optimal temperature and pH, possibly downregulated by phosphorylation and dependent on potassium ions, although they do not induce dimerization. The kinetic studies establish a double role for Lerepo4 in the stability and the activity of the enzyme, an effect that appears to reside in the Dfrp domain.
Our results glimpse a parallelism between two different kinds of mutation, a mutation inside switch I and the deletion of the TGS domain. Both mutants dampen their activity and improve potassium binding, indicating that they are affecting catalysis through switch I. Lerepo4 interaction gets to rescue the T100D mutant to almost normal values but not the truncated variant, pointing to TGS as a regulatory domain. Lerepo4 could be capable of affecting Drg1 activity indirectly by binding to the TGS domain, transducing the signal to the G domain, and also directly through contacts between its C-terminal part and switch I, as is hinted at by the solved structure from the yeast homologs (Fig. 4).
In short, we have clarified to some extent the catalytic features of Drg1, and its interaction with Lerepo4, foreseeing a paramount involvement of Lerepo4 in the task performed by Drg1, apart from its already proved protection against ubiquitination and its presence together with Drg1 in the ribosomes.
It would be interesting to determine the crystal structure of Drg1 in complex with a GTP analog to validate the experimental results shown about the architecture of the binding site. Efforts in this direction are presently under way in our laboratory.